| Background and ObjectivesGestational diabetes mellitus(GDM)is an abnormal glucose metabolism which was first developed or detected during pregnancy,with a worldwide incidence of 6.1%-15.2%,and has become one of the most common complications of pregnancy.In recent years,with the development China’s economy,the improvement of people’s living standards and the increase of elderly pregnant women after the liberalization of the two-child policy,the incidence of GDM has also shown an upward trend year by year.GDM can cause many short-term and long-term adverse effects on mothers and fetuses,among which,GDM-mediated fetal lung underdevelopment has become one of the important factors leading to severe respiratory diseases in neonates and young children,and it is particularly important to clarify its pathogenic mechanism.As a natural interface between mother and fetus,the placenta synthesizes and secretes numerous hormones,cytokines and chemical substances,which play an important role in maintaining the normal growth and development of the fetus.When patients suffer from gestational diabetes mellitus,affected by the high glucose and other adverse intrauterine environmental stimuli,the placenta will make adaptive changes to participate in pathophysiological processes such as inflammatory response,oxidative stress,and endocrine response,thereby affecting the fetus normal growth and development.Previous studies have confirmed the decisive role of placental dysfunction in neonatal lung underdevelopment,but the role of abnormal placenta in GDM-mediated fetal lung underdevelopment,especially the role of trophoblast,has not been fully elucidated.Exosomes,an important carrier of information communication between cells,have attracted increasing attention in normal physiological and pathological processes.They can carry various genetic materials and active molecules of parental cells to recipient cells,thereby regulating the gene expression of recipient cells and affecting their biological functions.In recent years,placental trophoblast-derived exosomes have been shown to play an important role in both normal pregnancy and pathological pregnancies,but most studies mainly focus on maternal cells such as vascular endothelial cells,smooth muscle cells,monocytes,etc.and there is no studies focusing on the existence of placental trophoblast-derived exosomes in fetal blood circulation and the regulation of placental trophoblast-derived exosomes on fetal development,especially the lung development.MicroRNA(miRNA)is a highly conserved non-coding RNA composed of about 22 nucleotides,which can inhibit the expression of mRNA by directly binding to the 3’untranslated region(3’UTR),thereby affecting the biological functions of target cells.As the main component of exosome contents,exosomal-miRNA mediated intercellular communication has been proved to be of great significance in the occurrence and development of various diseases,and a large number of studies have confirmed that miRNA had a vital role in fetal lung development.However,the role of miRNAs derived from placental trophoblast exosomes in fetal lung development has not yet been clarified.Therefore,from the perspective of "placenta-fetal lung" communication mediated by placental trophoblast exosomes in umbilical cord blood plasma,we first isolate and purify placental trophoblast-derived exosomes in umbilical cord blood plasma in GDM patients and trophoblast-derived exosomes by constructing GDM trophoblasts in vitro.Then we aim to systematically observe the role of GDM-associated exosomes in fetal lung underdevelopment by establishing A549 human alveolar epithelial cell in vitro model,embryonic lung explant ex vitro model and exosome intra-amniotic injection of pregnant mice in vivo model.Then the differentially expressed miRNAs were screened for verification through high-throughput sequencing technology and qRT-PCR.We will further explore the molecular mechanisms of the effects of placental trophoblast-derived exosomes on fetal lung development by constructing miRNA overexpression exosomes,which will provide new targets and ideas for its prevention and treatment.Part 1 Isolation and characterization of placental trophoblast-derived exosomes from umbilical cord blood plasma and trophoblast-derived exosomes from in vitro trophoblast modelsMethods:1.Placental trophoblast-derived exosomes from cord blood plasma were extracted using sucrose density gradient centrifugation combined with ultracentrifugation.2.GDM trophoblast models was constructed with or with 25 mM high glucose for HTR8/SVneo culture medium,and the cell culture supernatant was collected and ultracentrifuged to extract exosomes.3.Transmission electron microscopy(TEM),nanoparticle tracing technology(NTA)and Western Blot were used to charasterize exosomes.Results:1.Placental trophoblast-derived exosomes from normal and GDM umbilical cord blood plasma(NUB-exos and GDMUB-exos)and normal and GDM trophoblast-derived exosomes(NC-exos and HG-exos)were successfully isolated.2.All exosomes in the above groups showed typical cup-shaped bilayer membrane structures whose diameter ranging from 30 to 200nm.The WB results showed that they are positive for exosome marker proteins CD63,TSG101 and placenta-specific protein PLAP,and the NTA results showed that the concentrations of GDMUB-exos and HG-exos were higher than those of NUB-exos and NC-exos,respectively.ConclusionPlacental trophoblast-derived exosomes are present in cord blood plasma,and both of the clinical samples and in vitro GDM trophoblast models have demonstrated that high glucose could promotes exosome production and release.Part 2 The effects of GDM placental trophoblast-derived exosomes on fetal lung underdevelopmentMethods:1.A549 in vitro models:Firstly,the exosomes labeled with PKH67 fluorescent dye were used to evaluate the exosomes can be internalized by A549 cells.Then,cell viability was detected by CCK-8 assays,cell proliferation rates were detected by EdU cell proliferation assay,apoptotic rates was detected by flow cytometry,and Western Blot and qRT-PCR were used to detect the expression of lung surfactant-related proteins SPA,SPB,SPC and SPD(surfacant proteins A,B,C,and D)after treatment with different exosomes.In addition,the expression apoptosis-associated proteins Bax,Bcl2,Bim and Cleaved-caspase 3 were detected by Western Blot.2.Fetal lung explants ex vivo models:Firstly,E11.5 lung explants were isolate and culture with different groups of exosomes for 72 hours ex vitro,and the growth and of the lung explant were evaluated by counting the number of terminal buds and surface area of the lung explant through photography.After collecting the lung explants,fixed sections were used to detect changes in the proliferation and apoptosis cell ratio via EdU and Tunel experiments.Western Blot assays were used to detect the expression of SOX9,lung surfactant proteins A,B,C and D(SPA,SPB,SPC and SPD),and apoptosis-related proteins Bax,Bcl2,Bim and Cleaved-caspase 3 after different exosome treatments.qRT-PCR was used to detect the expression changes of surfactant proteins A,B,C and D and lung development-related genes Fgf10,VEGFa,Kdr and Flt-1 after different exosome treatments.Finally,tissue section immunofluorescence staining was used to further detect the expression changes of surfactant protein C(SPC)related to type Ⅱ alveolar epithelial cell differentiation.3.Exosmes intra-amniotic injection in vivo models:Firstly,different groups of exosomes were injected into the amniotic cavity of pregnant mice at E14.5.Then,at E18.5,the mice were dissected to collect the fetuses,and their body weight and crown-rump length were recorded.The fetal lung tissues were dissected,fixed,and sectioned for HE staining.The radial alveolar count(RAC)and lung surface tissue density were evaluated to assess fetal lung development.Western Blot was used to detect changes in the expression of surfactant proteins A,B,C and D(SPA,SPB,SPC and SPD),and apoptosis-related proteins Bax,Bcl2,Bim and Cleaved-caspase 3 after different exosomes treatments.qRT-PCR was used to detect the expression changes of surfactant proteins A,B,C and D and lung development and maturation-related genes BMP2,BMP4 and ID1 after different exosomes treatments.EdU and Tunel experiments were conducted on fetal lung tissue sections to detect changes in the proliferation and apoptosis cell ratio.Finally,tissue section immunofluorescence staining was used to further detect the expression changes of surfactant protein C(SPC)related to type Ⅱalveolar epithelial cell differentiation.Results:1.In A549 in vitro models,all groups of PKH67-labeled exosomes were internalized by A549 cells.Compared to the NUB-exos and NC-exos groups respectively,A549 cells treated with GDMUB-exos and HG-exos showed reduced cell viability and proliferation and an increased apoptosis rate.Western Blot and qRT-PCR results showed a decrease in the expression of surfactant proteins A,B,C and D(SPA,SPB,SPC,and SPD)and anti-apoptotic protein Bcl2,as well as an increase in the expression of pro-apoptotic proteins Bax,Bim,and Cleaved-caspase2.In lung explant in vitro models,compared to the NUB-exos and NC-exos groups respectively,the number of terminal buds of lung explants significantly decreased accompanied by lung surface area reduction after treatment with GDMUB-exos and HG-exos.EdU and Tunel experiments showed a decrease in the proliferation cell ratio and an increase in the apoptosis cell ratio.Western Blot and qRT-PCR results showed a significant decrease in the transcriptional and post-transcriptional levels of surfactant proteins A,B,C,and D,as well as a decrease in the expression of anti-apoptotic protein Bcl2 and an increase in the expression of pro-apoptotic proteins Bax,Bim,and Cleaved-caspase 3.Additionally,the expression of key genes related to lung development,including Fgf10,VEGFa,Kdr,and Flt-1,was significantly reduced.Finally,tissue section immunofluorescence staining showed a decrease in the expression of surfactant protein C(SPC).3.In exosmes intra-amniotic injection in vivo models,compared to the NUB-exos and NC-exos groups,treatment with GDMUB-exos and HG-exos resulted in a significant decrease in fetal body weight and crown-rump length.HE staining showed a decrease in the radial alveolar count(RAC)and an increase in lung surface tissue density.Western Blot and qRT-PCR results showed a significant decrease in the transcriptional and post-transcriptional levels of surfactant proteins A,B,C,and D,as well as a decrease in the expression of anti-apoptotic protein Bcl2 and an increase in the expression of pro-apoptotic proteins Bax,Bim,and Cleaved-caspase 3.Additionally,the expression of genes related to lung development and maturation,including BMP2,BMP4,and ID1,was significantly reduced.Finally,tissue section immunofluorescence staining showed a decrease in the expression of surfactant protein C(SPC).Conclusion:GDM placental trophoblasts-derived exosoms can disrupt the homeostasis of lung epithelial cells,inhibit proliferation,promote apoptosis,reduce surfactant protein expression,damage lung branching morphogenesis,delay fetal lung development,and lead to fetal lung underdevelopment.Part 3:Screening and validation of differentially expressed miRNAs in placental trophoblast-derived exosomes from umbilical cord blood plasma of gestational diabetes mellitus(GDM)patientsMethods:1.High-throughput sequencing technology was applied to analyze the miRNA expression profiles of placental-derived exosomes in umbilical cord blood plasma from normal and GDM patients and to screen for differentially expressed miRNAs.2.TargetScan and miRanda online websites were used to predict target genes for the differentially expressed miRNAs,and gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed on these target genes.3.Differential expression of miRNAs in placental trophoblast-derived exosomes was validated using qRT-PCR.Results:1.A total of 1885 miRNAs were detected,among which 55 miRNAs were differentially expressed(10 miRNAs upregulated and 45 miRNAs downregulated).2.Target gene prediction was performed for the differentially expressed miRNAs,and GO enrichment analysis of the predicted target genes revealed that they were mainly enriched in signaling pathways such as protein binding,transferase activity,ATP binding,and cell cycle.KEGG analysis revealed that the target genes were mainly enriched in the PI3K-AKT signaling pathway,cancer pathway,calcium signaling pathway,Rabl pathway,and Hippo pathway.3.qRT-PCR assays confirmed miR-185-5p as the next research target.Conclusion:The miRNA expression profiles of placental-derived exosomes in umbilical cord blood plasma from normal and GDM patients are different.Part 4:The mechanism of miR-185-5p from GDM placental trophoblast-derived exosomes mediated fetal lung underdevelopmentMethods:1.Stably overexpressing HEK-293T cell lines with miR-185-5p were constructed using a lentiviral vector.Cell culture supernatant was collected,and exosomes were isolated using ultracentrifugation.The exosomes were characterized using TEM,NTA,and Western Blot.qRT-PCR was used to detect the expression of miR-185-5p in the 293T cell lysates and in the exosomes from the cell culture supernatant.2.In A549 in vitro models:Exosome internalization experiments were conducted with PKH67-labeled exosomes to observe whether exosomes could be internalized by A549 cells.Cell viability was assessed using the CCK-8 assay,cell proliferation was assessed using the EdU cell proliferation assay,apoptosis was assessed using flow cytometry,and Western Blot was used to detect changes in the expression of lung surfactant proteins SPA,SPB,SPC,and SPD after treatment with different exosomes.3.In lung explants ex vivo models:E11.5 pregnant mouse embryos were dissected,and the lung explants were cultured ex vitro for 72 hours.Exosomes from different groups were added during the culture process.The growth and development of the fetal lung explants were evaluated by calculating the number of terminal buds and surface area of the explants based on photographs taken during the culture period.EdU and Tunel experiments were conducted to assess changes in the proportion of proliferating and apoptotic cells in the lung explants after collection.Western Blot was used to detect changes in the expression of lung surfactant proteins SPA,SPB,SPC,and SPD after treatment with different exosomes.4:In exosomes intra-amniotic injection in vivo models.Different groups of exosomes were injected into the amniotic cavity of E14.5 pregnant mice.Pregnant mice were then sacrificed by caesarean section at E18.5 to collect fetuses.Lung tissue from the fetuses was dissected and fixed,and HE staining was performed.Radial alveolar count(RAC)and lung surface tissue density were used to assess fetal lung development.EdU and Tunel experiments were conducted to assess changes in the proportion of proliferating and apoptotic cells in the lungs after collection.Western Blot was used to detect changes in the expression of surfactant proteins SPA,SPB,SPC,and SPD.5.TargetScan and miRanda were used to predict miR-185-5p target genes,and a dual luciferase reporter gene assay was used to further verify the targeted regulation relationship between miR-185-5p and SOX13/Soxl3.Western Blot were used to detect changes in the expression of SOX13/Sox13 and β-catenin pathway proteins.Results:1.We successfully established HEK-293T cell lines overexpressing miR-185-5p using a lentiviral vector and extracted exosomes from them.Both groups of exosomes exhibited the typical cup-shaped double membrane structure with diameters ranging from 30-200nm,and Western Blot results indicated the expression of the exosome marker proteins CD63,TSG101,CD9,while the endoplasmic reticulum protein Calnexin was not expressed.qRT-PCR results showed a significant increase in the expression of miR-185-5p in both the 293T cells and the exosomes from the cell culture supernatant.2.In A549 in vitro models:both groups of PKH67-labeled exosomes were able to be internalized by the A549 cells.Compared to the control group,the addition of miR-185-5p overexpressing exosomes resulted in decreased cell viability and proliferation,and increased apoptosis.Western Blot results showed decreased expression of surfactant proteins SPA,SPB,SPC,and SPD.3.In lung explant ex vivo models,compared to the control group,the addition of miR-185-5p overexpressing exosomes resulted in a significant decrease in the number of terminal buds and surface area of the fetal lung explants.Edu and Tunel experiments showed a decrease in the proportion of proliferating cells and an increase in the proportion of apoptotic cells.Western Blot showed decreased expression of surfactant proteins SPA,SPB,SPC,and SPD.4.In exosomes intra-amniotic injection in vivo models:c HE staining results showed a decrease in radial alveolar count(RAC)and an increase in lung surface tissue density.Edu and Tunel experiments showed a decrease in the proportion of proliferating cells and an increase in the proportion of apoptotic cells.Western Blot showed decreased expression of surfactant proteins SPA,SPB,SPC,and SPD.5.TargetScan and miRDB predicted SOX13/Sox13 as the homologous target gene of miR-185-5p in humans and mice,and a dual luciferase reporter gene assay confirmed the binding site between them.Western Blot showed a significant decrease in the expression of SOX13/Sox13 and β-catenin after treatment with miR-185-5p overexpressing exosomes.Conclusion:miR-185-5p derived from GDM placental trophoblast-derived exosomes can mediate the occurrence of fetal lung underdevelopment through SOX13(Sox13)/β-catenin pathway. |
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